Traditional Chinese medicine composition for diabetic foot ulcer and preparation method thereof

Abstract

The invention discloses a traditional Chinese medicine composition for diabetic foot ulcer and a preparation method thereof. Comprising effective extracts of folium ginkgo, radix astragali, rhizoma corydalis and rhizoma anemones raddeanae as active pharmaceutical ingredients. The traditional Chinese medicine composition has an obvious improvement effect on diabetic foot ulcer, the cure rate of a rat with the diabetic foot ulcer can be obviously increased, wound healing is accelerated, and the action mechanism of the traditional Chinese medicine composition is closely related to a Wnt / beta-catenin signal channel. In addition, the traditional Chinese medicine composition provided by the invention also has the advantages of simplicity in preparation, low cost, convenience in carrying and remarkable effect.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and in particular relates to a traditional Chinese medicine composition for treating diabetic foot ulcers and a preparation method thereof. Background Art

[0002] Diabetic foot (DFU) is a common complication of diabetes. Distal lower limb nerve abnormalities and varying degrees of vascular disease can lead to foot infections, ulcers, and / or deep tissue destruction. Mild cases can cause foot ulceration, while severe cases can develop gangrene and even amputation. The incidence of DFU in diabetic patients can reach 15%, making it a major cause of disability and mortality among patients with diabetes and a serious social threat. The development of new medium- and long-acting drugs with high drug loading, excellent efficacy, minimal side effects, and ease of use is urgent. Traditional Chinese medicine (TCM), with its multi-component, multi-target, and strong synergistic effects, offers unique advantages in treating the complexities of DFU. For example, modified Taohong Siwu Decoction (Taohong Siwu Decoction) and Honglingdan Ointment (Honglingdan Ointment) demonstrate promising therapeutic effects for DFU. Both TCM theory and modern pharmaceutical research demonstrate that Ginkgo biloba can promote blood circulation, remove blood stasis, dredge meridians, relieve pain, and improve microvascular circulation. Two-headed scutellaria baicalensis (Liangtoujian) can dispel rheumatism, reduce carbuncles, and treat carbuncle ulcers. Astragalus root (Huangqi) can expel toxins, drain pus, and promote tissue regeneration. Corydalis yanhusuo can promote qi and blood circulation and has an analgesic effect. These four Chinese medicines play an important role in the treatment of diabetic foot ulcers in different directions. Summary of the Invention

[0003] The purpose of the present invention is to provide a traditional Chinese medicine composition for diabetic foot ulcers and a preparation method thereof, wherein the traditional Chinese medicine composition has the advantages of simple preparation, low cost, easy to carry and significant effect.

[0004] The technical solution adopted by the present invention is as follows:

[0005] A traditional Chinese medicine composition for diabetic foot ulcer and a preparation method thereof, comprising extracts of ginkgo biloba leaves, astragalus root, yanhusuo root and liangtoujian.

[0006] Preferably, in parts by weight, the raw materials include 10-25 parts by weight of Ginkgo biloba extract, 8-15 parts by weight of Astragalus root extract, 5-20 parts by weight of Corydalis yanhusuo extract and 10-15 parts by weight of Rhizoma Coptidis extract.

[0007] Preferably, by weight, the raw materials include 15 parts by weight of Ginkgo biloba extract, 10 parts by weight of Astragalus root extract, 8 parts by weight of Corydalis yanhusuo extract and 15 parts by weight of Rhizoma Coptidis extract.

[0008] In the above scheme, the ginkgo leaves, astragalus, yanhusuo and liangtoujian are all Chinese medicinal materials.

[0009] Preferably, the Chinese medicine composition is prepared by the following method: clean the ginkgo leaves, dry them at 50-80℃, crush them into 100-10 mesh powder, extract them with 5-15 times the volume of 40-95% ethanol under reflux for 1-3 times, each time for 1-3 hours, combine the extracts, recover the extracts, dry them below 80℃, grind them into fine powder for later use; clean the astragalus root, dry them at 50-80℃, crush them into 100-10 mesh powder or granules, extract them with 5-15 times the volume of distilled water under ultrasonication for 1-3 times, each time for 0.5-3 hours, combine the extracts, recover the extracts, dry them below 80℃, grind them into fine powder for later use; clean the corydalis root, dry them at 50 -80 ℃ drying, crushing into 100 mesh-10 mesh powder, heating and refluxing extraction with 5-15 times the volume of 40-95% ethanol for 1-3 times, each time for 1-3 hours, combining the extracts, recovering the extracts, drying below 80 ℃, grinding into fine powder for later use; cleaning the two ends, drying at 50-80 ℃, crushing into 100 mesh-10 mesh powder or granules, adding 8-10 times the volume of 50-95% ethanol for ultrasonic extraction for 1-3 times, each time for 0.5-3 hours, combining the extracts, recovering the extracts, drying below 80 ℃, grinding into fine powder for later use; mixing the obtained medicinal material extracts in proportion to obtain the traditional Chinese medicine composition of the present invention;

[0010] The Chinese medicine composition of the present invention is a common external preparation, such as powder, patch, gel, and gelatin sponge.

[0011] The present invention has the following beneficial effects: The traditional Chinese medicine composition has a significant improvement effect on diabetic foot ulcers, significantly accelerating the cure rate and wound healing in rats with diabetic foot ulcers. Its mechanism of action is closely related to the Wnt / β-catenin signaling pathway. Furthermore, the traditional Chinese medicine composition provided by the present invention is simple to prepare, low in cost, easy to carry, and has significant effects.

[0012] Specific implementation: The present invention is described in detail below with reference to specific embodiments, but is not intended to limit the present invention.

[0013] Example 1 (Gelatin sponge composition)

[0014] 1. Prescription raw materials: raw materials Ginkgo biloba extract 15g, Astragalus root extract 10g, Corydalis yanhusuo extract 8g and Rhizoma Coptidis extract 15g.

[0015] 2. Preparation method: Add formaldehyde solution to purified water to make a 10% formaldehyde solution, soak the crushed gelatin particles in the raw material pure water solution, heat the gelatin particles after they fully absorb water and expand, control the temperature at 35°C, and continuously stir to dissolve, then cool them to 15°C, stir them, and continue to add formaldehyde solution, continue stirring to obtain gelatin foam, freeze and dry the obtained gelatin foam to obtain a drug-loaded gelatin sponge preparation.

[0016] Efficacy trials

[0017] 1. Materials and Methods

[0018] 1.1 Experimental Materials

[0019] Male rats were purchased from Changchun Yisi Animal Center; blood glucose meter was purchased from Abbott Laboratories, USA; streptozotocin was purchased from Sigma, USA; and human epidermal growth factor was purchased from Shenzhen Huashengyuan Gene Engineering Development Co., Ltd.

[0020] 1.2 Preparation and grouping of diabetic foot animal models

[0021] 90 male SD rats of SPF grade and weighing 200±20g were purchased. Ten rats in the normal control group were fed with maintenance feed, and the remaining 80 SD rats were fed with high-sugar and high-fat feed for 6 weeks. Six weeks later, the model group was injected with streptozotocin. The SD rats were fasted for 12 hours before injection but not water. The rats were weighed and injected intraperitoneally. STZ solution was injected rapidly at a dose of 30mg / kg to establish the model. The normal control group was injected with citric acid-sodium citrate buffer at a dose of 30mg / kg. After 72 hours, blood was collected from the tail vein to test the random blood glucose of the rats. Thereafter, random blood glucose was tested with a blood glucose meter every 3 days, and the rats were weighed at the same time. If the blood glucose concentration was ≥16.7mmol / L for three consecutive times, and there were symptoms of "three more and one less" and yellowing of hair, it meant that the diabetic rat model was successfully established.

[0022] After maintaining hyperglycemia in diabetic rats for one week, all rats were anesthetized with isoflurane inhalation. After satisfactory anesthesia, the rat feet were routinely disinfected, and a full-thickness skin defect trauma model was created on the rat feet using surgical scissors. The diabetic foot rats with the constructed model were divided into three groups: a blank control group, a positive drug group (human epidermal growth factor), and a drug group, with 20 rats in each group.

[0023] 1.3 Treatment methods and experimental observation indicators

[0024] The foot of the drug group was bandaged with gelatin sponge containing a traditional Chinese medicine composition and sterile gauze; the positive drug group was treated with a topical solution of recombinant human epidermal growth factor, followed by an external bandage with sterile gauze; the blank control group was bandaged with gelatin sponge and sterile gauze without the active ingredient of the traditional Chinese medicine. Because the blank and drug groups are self-degradable and ultimately completely absorbed by the body, the dressing change interval was determined based on the degradation cycle, with a dressing change every 7 days. The control group was changed every 2 days for 28 consecutive days. On the 7th, 14th, and 28th day after modeling, the wound size was measured, the wound healing rate was calculated, and tissues including the wound and periwound skin were excised for histopathological examination and the levels of various test indicators. The wounds were observed until healing was complete, and the wound healing time was calculated.

[0025] 1.4 RealTime-qPCR Experimental Method

[0026] 1.4.1 RNA extraction

[0027] Weigh 50 mg of tissue, mince it with scissors and transfer it to a new EP tube; add 1 ml of Trizol to the tissue, grind the tissue evenly with a grinder, and let it stand at room temperature for 5 minutes; add 200 μL of chloroform, shake gently for 15 seconds (to mix well), let it stand for 10 minutes, and centrifuge at 12,000 rpm and 4°C for 15 minutes; transfer the supernatant to a new EP tube, add an equal volume of isopropanol, ice bath for 10 minutes, and centrifuge at 12,000 rpm and 4°C for 10 minutes; remove the supernatant, add 1 ml of 75% ethanol (pre-cooled) and wash twice, shake gently without blowing it away, and centrifuge at 12,000 rpm and 4°C for 5 minutes; remove the supernatant, dry it, add DEPC water (20-50 μl), and store it at -80°C.

[0028] 1.4.2 First-strand cDNA synthesis

[0029] (1) Removal of residual genomic DNA: Remove RNA from the -80°C freezer, thaw at 4°C, prepare the following mixture in an RNase-free centrifuge tube, and mix thoroughly by gently pipetting. Incubate at 42°C for 2 min.

[0030]

[0031] (2) Preparation of reverse transcription reaction system (20 μL system): directly add 2×Hifair to the reaction tube of (1) “residual genomic DNA removal” ⅡSuperMix plus, mix gently by pipetting.

[0032]

[0033] (3) Reverse transcription program setting: Prepare the reverse transcription reaction system (20 μL system) and incubate the above mixed solution according to the program in the table below.

[0034]

[0035] (4) SYBR Green qPCR: Prepare the reaction system on ice as shown in Table 1-1 and Table 1-2:

[0036] Table 1-1 Reaction system prepared on ice

[0037]

[0038] Table 1-2 Circulation system

[0039]

[0040] Incubate the above mixture according to the procedure in the table below.

[0041] (5) Primer sequence: Primer information is shown in Table 1-3.

[0042] Table 1-3 Primer information

[0043]

[0044] 1.5 Statistical analysis

[0045] SPSS 21.0 software was used to analyze the data. Data with normal distribution and homogeneity of variance were analyzed using t-test or analysis of variance, and p < 0.05 was considered statistically significant.

[0046] 2 Experimental results and analysis

[0047] 2.1 Body weight measurement results of rats in each group

[0048] The body weight measurements for the blank and model groups are shown in Table 2-1. There was no significant difference in body weight between the two groups before modeling (P>0.05). After modeling, the body weight of the model group rats increased significantly. After 35 days, the body weight of the model group rats decreased. Following STZ injection, the body weight of the model group rats gradually decreased.

[0049] Table 2-1 Results of rat body weight measurement

[0050]

[0051] 2.2 Blood Glucose Measurement Results for Each Group of Rats: Blood glucose measurement results for the blank and model groups are shown in Table 2-2. There was no significant difference in blood glucose between the two groups before modeling (P>0.05). After 42 days, blood glucose levels in the model group increased. Following STZ injection, blood glucose levels in the model group gradually increased, reaching a random blood glucose level exceeding 16.7 mmol / L, significantly higher than that of the normal control rats (P<0.01), indicating successful diabetic model establishment.

[0052] Table 2-2 Blood glucose measurement results of rats

[0053]

[0054] 2.3 Results of the cure rate measurements of rats in each group: The results of the cure rate measurements of rats in each group are shown in Table 2-3. Compared with the normal group, the 21-day cure rate of mice in the model group was significantly lower (P<0.05). Compared with the model group, the 21-day cure rate of mice in the drug group was significantly higher (P<0.01). These results indicate that the drug has a significant healing effect on diabetic foot.

[0055] Table 2-3 Cure rate of rats in each group

[0056]

[0057] Note: *Compared with the normal group, *P<0.05, **P<0.01; #Compared with the model group, #P<0.05, ##P value<0.01

[0058] 2.4 Wound Healing Time Measurement Results for Each Group of Rats: The wound healing time measurement results for each group of rats are shown in Table 2-4. Compared with the normal group, the average wound healing time of the model group was significantly increased (P < 0.01). Compared with the model group, the average wound healing time of the drug group was significantly decreased (P < 0.05). These results indicate that the drug has a significant effect on the healing of diabetic foot wounds.

[0059] Table 2-4 Measurement results of wound healing time of rats in each group

[0060]

[0061] Note: *Compared with the normal group, *P<0.05, **P<0.01; #Compared with the model group, #P<0.05, ##P value<0.01

[0062] 2.5 Effects of drugs on the Wnt / β-catenin pathway

[0063] 2.5.1 Effects of Drugs on Wnt1 and Wnt3a mRNA Expression: Real-time qPCR results for the effects of drugs on Wnt1 and wnt3a mRNA expression are shown in Tables 2-5 and 2-6. Regarding Wnt1 mRNA expression in wound tissue, after wound modeling, the model group showed significantly lower Wnt1 mRNA expression compared to the normal group (P < 0.01). Compared with the model group, Wnt1 mRNA expression in the drug treatment groups increased significantly in all groups except the medium and low-dose drug groups on day 21 (P < 0.05). Regarding Wnt3a mRNA expression in wound tissue, after wound modeling, the model group showed significantly lower Wnt3a mRNA expression compared to the normal group (P < 0.01). Compared with the model group, Wnt1 mRNA expression in the drug intervention groups increased significantly in all other groups (P < 0.05).

[0064] Table 2-5 Comparison of relative expression of Wnt1 mRNA in wound tissues of rats in each group

[0065]

[0066] Note: *Compared with the normal group, *P<0.05, **P<0.01; #Compared with the model group, #P<0.05, ##P value<0.01

[0067] Table 2-6 Comparison of relative expression of wnt3a mRNA in wound tissue of rats in each group

[0068]

[0069] Note: *Compared with the normal group, *P<0.05, **P<0.01; #Compared with the model group, #P<0.05, ##P value<0.01

[0070] 2.5.2 Expression of β-Catenin mRNA in wound tissue of rats in each group at different time points: The Real Time-q PCR results of the effects of drugs on β-Catenin mRNA expression are shown in Table 2-7. After wound modeling, the expression of β-Catenin mRNA in wound tissue of the model group was significantly lower than that in the normal wound group (P<0.01). Compared with the model group, the expression of β-Catenin mRNA in the high and medium drug treatment groups was significantly increased (P<0.05).

[0071] Table 2-7 Comparison of relative expression of β-Catenin mRNA in wound tissues of rats in each group

[0072]

[0073] Note: *Compared with the normal group, *P<0.05, **P<0.01; #Compared with the model group, #P<0.05, ##P value<0.01

[0074] 2.5.3 Expression of GSK3β mRNA in wound tissue of rats in each group at different time points: Real Time-q PCR results of the effects of drugs on GSK3β mRNA expression are shown in Table 2-8. After wound modeling, the expression of GSK3β mRNA in wound tissue of the model group was significantly higher than that in the normal group (P<0.01); compared with the model group, the expression of GSK3β mRNA in the drug intervention group was significantly decreased (P<0.05).

[0075] Table 2-8 Comparison of relative expression of GSK3β mRNA in wound tissues of rats in each group

[0076]

[0077] Note: *Compared with the normal group, *P<0.05, **P<0.01; #Compared with the model group, #P<0.05, ##P value<0.01

[0078] Research suggests that the Wnt / β-catenin pathway plays a key role in the granulation tissue proliferation phase of diabetic foot ulcer healing. The healing mechanism of diabetic foot ulcers is closely related to the regulation of the Wnt / β-catenin signaling pathway. This study examined the protein expression levels of β-catenin, glycogen synthase kinase-3β (GSK-3β), and R-spondin 3 (RSPO-3), key factors in the Wnt signaling pathway. β-catenin and GSK-3β are key downstream proteins in this pathway. β-catenin plays a promoting role in wound healing; high expression of both promotes cell proliferation and differentiation in wound tissue, leading to the growth of granulation tissue. GSK-3β, on the other hand, plays an inhibitory role in wound healing, being expressed at a low level during periods of cell proliferation, differentiation, and granulation tissue growth. Wnt1 and Wnt3a are the main Wnt proteins that activate the canonical Wnt pathway and participate in the Wnt canonical signaling pathway. Studies have found that they have the function of regulating the development of the skin and its appendages, promoting wound angiogenesis and epithelial remodeling, and thus participate in the process of wound repair and healing.

[0079] 3 Conclusion

[0080] The research was conducted by constructing a rat model of diabetic foot ulcer. The results showed that the drug in this study can be used in rats with diabetic foot ulcers, and can accelerate the cure rate of foot ulcers in diabetic rats and accelerate wound healing. Its mechanism of action is closely related to the Wnt / β-catenin signaling pathway.

Claims

1. A Chinese medicine composition for diabetic foot ulcer and a preparation method thereof, characterized in that: The traditional Chinese medicine composition comprises the following components: 10 to 25 parts by weight of ginkgo leaf extract, 8 to 15 parts by weight of astragalus extract, 5 to 20 parts by weight of Corydalis yanhusuo extract and 10 to 15 parts by weight of liangtoujian extract.

2. The Chinese medicine composition according to claim 1, characterized in that The traditional Chinese medicine composition comprises the following components: 15 parts by weight of ginkgo leaf extract, 10 parts by weight of astragalus extract, 8 parts by weight of Corydalis yanhusuo extract and 15 parts by weight of Rhizoma Coptidis extract.

3. A method for preparing the Chinese medicine composition according to any one of claims 1 to 2, characterized in that: The preparation method is prepared by the following steps: (1) Clean the ginkgo leaves, dry them at 50-80°C, grind them into 100-10 mesh powder, and extract them with 5-15 times the volume of 40-95% ethanol under heating and refluxing for 1-3 times, each time for 1-3 hours. Combine the extracts, recover the extracts, dry them below 80°C, grind them into fine powder, and set aside. (2) Clean the Astragalus root, dry it at 50-80°C, crush it into 100-10 mesh powder or granules, and extract it with 5-15 times the volume of distilled water by ultrasonic extraction for 1-3 times, each time for 0.5-3 hours. Combine the extracts, recover the extracts, dry them below 80°C, grind them into fine powder, and set aside. (3) Clean the Corydalis yanhusuo, dry it at 50-80°C, grind it into a 100-10 mesh powder, and extract it with 5-15 times the volume of 40-95% ethanol under heating and refluxing for 1-3 times, each time for 1-3 hours. Combine the extracts, recover the extracts, dry them below 80°C, grind them into fine powder, and set aside. (4) Clean the two tips, dry at 50-80°C, crush into 100-10 mesh powder or granules, add 8-10 times the amount of 50-95% ethanol and ultrasonically extract 1-3 times, each time for 0.5-3 hours, combine the extracts, recover the extracts, dry below 80°C, grind into fine powder, and set aside; (5) The medicinal material extracts obtained in steps (1-4) are mixed uniformly according to the proportions to obtain the Chinese medicine composition of the present invention; (6) Using the composition obtained in step (5) as a raw material, an external preparation is prepared according to a conventional preparation method.

4. A preparation for external use, characterized in that The external preparation contains the traditional Chinese medicine composition according to any one of claims 1 to 2.

5. The external preparation according to claim 4, characterized in that The external preparation is any one of a powder, a patch, a gel, and a gelatin sponge.

6. The external preparation according to claims 4 and 5, characterized in that The external preparation is prepared according to the method according to claim 3.

7. The Chinese medicine composition according to any one of claims 1 to 2 and the external preparation according to any one of claims 4 to 6 are used for diabetic foot ulcer disease.

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